Methyl modifications on mRNA are essential for mammalian cell fate decisions and have recently been shown to play important roles in the progression of many human cancers. The demethylase FTO, which erases abundant N6-methyladenosine modifications to dynamically regulate mRNA methylation, is linked to initiation and progression of cancers including brain, breast, gastric, cervical, mesothelioma, and leukemia. Recent work has shown that inhibition of FTO suppresses tumor progression in glioblastoma, the most common and deadliest form of brain cancer, suggesting FTO and other mRNA-modifying enzymes may be promising anti-cancer drug targets. However, we lack a mechanistic understanding of how FTO targets its modified mRNA substrates, impacts mRNA function, and contributes to disease progression. Our long-term goal is to define the biochemical, structural, and molecular mechanisms that regulate mRNA demethylation in the cell, and to develop the new tools and understanding needed to characterize demethylation activity across the transcriptome and in diverse human disease states. The work outlined in this proposal will: (1) define the structural and biochemical basis of FTO target selectivity at the atomic level using X-ray crystallography and enzymology with synthetic modified nucleotide analogs, (2) generate bioorthogonal chemical probes that covalently trap FTO-installed demethylation intermediates to directly map sites of demethylation on mRNA, and (3) explore the molecular mechanisms through which FTO-installed demethylation intermediates may directly regulate mRNA function. This work is impactful and significant because it will advance the field?s understanding of how methyl eraser FTO is targeted to different mRNA modifications and how unexplored, metastable intermediate modifications installed during FTO-mediated demethylation impact mRNA function. Furthermore, the tools we develop to trap demethylation intermediates will be critical to map sites of mRNA demethylation across the transcriptome in different cell types and disease states, which will allow our lab and others to directly assess the role of mRNA demethylation in human cancers linked to FTO function.